Medical prosthetic devices and implants having improved biocompatibility

ABSTRACT

Disclosed are medical prosthetic device or medical implants which exhibit improved biocompatibility. The devices or implants include a metal material, e.g., titanium, in which the metal surfaces are coated with a corresponding hydride material that contains one or more biomolecule substance. The biomolecule substance may contain one or more biologically active molecules, e.g., bio-adhesives, biopolymers, blood proteins, enzymes, extracellular matrix proteins, extracellular matrix biomolecules, growth factors and hormones, peptide hormones, deoxyribonucleic acids, ribonucleic acids, receptors, inhibitors, drugs, biologically active anions and cations; vitamins; adenosine monophosphate (AMP), adenosine diphosphate (ADP) or adenosine triphosphate (ATP), marker biomolecules, amino acids, fatty acids, nucleotides (RNA and DNA bases), or sugars.

RELATED APPLICATIONS

This application is a 37 C.F.R. § 1.53(b) divisional of U.S. applicationSer. No. 10/010,140 filed Dec. 6, 2001, which claims priority on U.S.Provisional Application 60/254,987 filed on Dec. 12, 2000, and DenmarkPatent Application No. PA 2000 01829 filed on Dec. 6, 2000. The entirecontents of each of these application is hereby incorporated herein byreference.

FIELD OF THE INVENTION

The present invention concerns medical prosthetic devices and implantshaving improved biocompatibility.

BACKGROUND OF THE INVENTION

It has been proposed to improve the biocompatibility of e.g., a titaniumprosthesis by coating metal surfaces thereof with a layer of titaniumhydride. Such a hydride layer may be applied by plasma bombardment, orin may be applied by electrolysis; see, for example. U.S. patentapplication Ser. No. 09/868,965, now U.S. Pat. No. 6,627,321, which ishereby incorporated by reference.

It has also been proposed to improve the biocompatibility of prosthesesor implants by binding or integrating various active biomolecules to thesurface of the prosthesis, e.g., on to the metallic surface of atitanium prosthesis. It has been the aim with implants prepared this waythat they have improved fit; exhibit increased tissue stickiness andincreased tissue compatibility; have a biologically active surface forincreased cell growth, differentiation and maturation; exhibit reducedimmunoreactivity; exhibit antimicrobial activity; exhibit increasedbiomineralisation capabilities; result in improved wound and/or bonehealing; lead to improved bone density; have reduced “time to load” andcause less inflammation.

Such binding has often been proposed carried out using for examplechemical reactants having two reactive functionalities such as formalinor glutaraldehyde, but the reactive nature of these agents often leadsto the biomolecules becoming biologically inactive and/or with enhancedimmunoreactivity which is undesirable.

SUMMARY OF THE INVENTION

It has now surprisingly been found that it is possible to interlock,bind, trap and/or integrate a wide variety of biomolecules in or with ahydride layer during the inorganic process of formation of such ahydride layer on metals by electrolysis. Prior to this observation, itwas considered very difficult to bind and stabilize unmodified,bioactive biomolecules on metals, especially for use as bioactivesurfaces on metals for use as implants in the vertebrate body in vivo.

The invention, therefore, concerns a medical prosthetic device orimplant containing a metal material (A) selected from the groupconsisting of titanium or an alloy thereof, zirconium or an alloythereof, tantalum or an alloy thereof, hafnium or an alloy thereof,niobium or an alloy thereof and a chromium-vanadium alloy, whereinsurface parts of the metal material (A) are coated with a layer of acorresponding hydride material (B) selected from titanium hydride,zirconium hydride, tantalum hydride, hafnium hydride, niobium hydrideand chromium and/or vanadium hydride, respectively, characterised inthat the layer of hydride material (B) comprises one or more biomoleculesubstances (C) associated therewith.

The invention further concerns a method for preparing a medicalprosthetic device or implant as defined above, said method comprisingsubjecting surface parts of the metal material (A) as defined above toan electrolysis treatment to form the layer of hydride material (B),said electrolysis treatment being carried out in the presence of one ormore biomolecule substances (C).

DETAILED DESCRIPTION OF THE INVENTION

In the present context, the phrase “medical prosthetic device andimplant” includes within its scope any device intended to be implantedinto the body of a vertebrate animal, in particular a mammal such as ahuman. Non-limiting examples of such devices are medical devices thatreplace anatomy or restore a function of the body, such as the femoralhip joint; the femoral head; acetabular cup; elbow, including stems,wedges, articular inserts; knee, including the femoral and tibialcomponents, stem, wedges, articular inserts or patellar components;shoulders including stem and head; wrist; ankles; hand; fingers; toes;vertebrae; spinal discs; artificial joints; dental implants;ossiculoplastic implants; middle ear implants including incus, malleus,stapes, incus-stapes, malleusincus, malleus-incus-stapes; cochlearimplants; orthopaedic fixation devices such as nails, screws, staplesand plates; heart valves; pacemakers; catheters; vessels; space fillingimplants; implants for retention of hearing aids; implants for externalfixation; and also intrauterine devices (IUDs); and bioelectronicdevices such as intracochlear or intracranial electronic devices.

In the present context, the term “biomolecule” is intended to cover andcomprise within its meaning a very wide variety of biologically activemolecules in the widest sense of the word, be they natural biomolecules(i.e., naturally occurring molecules derived from natural sources),synthetic biomolecules (i.e., naturally occurring molecules preparedsynthetically as well as non-naturally occurring molecules or forms ofmolecules prepared synthetically) or recombinant biomolecules (i.e.,prepared through the use of recombinant techniques).

A non-limiting list of main groups of and species biomolecules that arecontemplated as being suitable for incorporation into a metal hydridelayer (in a stable and/or physiologically reversible manner) inaccordance with the invention is given below.

Extracted Biomolecules

Bioadhesives:

These are biomolecules that mediate attachment of cells, tissue, organsor organisms onto non-biological surfaces like glass, rock etc. Thisgroup of bio-molecules includes the marine mussel adhesive proteins,fibrin-like proteins, spider-web proteins, plant-derived adhesives(resins), adhesives extracted from marine animals, and insect-derivedadhesives (like resilins). Some specific examples of adhesives are:

Fibrin; fibroin; Mytilus edulis foot protein (mefpl, “mussel adhesiveprotein”); other mussel's adhesive proteins; proteins and peptides withglycine-rich blocks; proteins and peptides with poly-alanine blocks; andsilks.

Cell Attachment Factors:

Cell attachment factors are biomolecules that mediate attachment andspreading of cells onto biological surfaces or other cells and tissues.This group of molecules typically contains molecules participating incell-matrix and cell-cell interaction during vertebrate development,neogenesis, regeneration and repair. Typical biomolecules in this classare molecules on the outer surface of cells like the CD class ofreceptors on white blood cells, immunoglobulins and haemagglutinatingproteins, and extracellular matrix molecules/ligands that adhere to suchcellular molecules. Typical examples of cell attachment factors withpotential for use as bioactive coating on metal hydride-coated implantsare: Ankyrins; cadherins (Calcium dependent adhesion molecules);connexins; dermatan sulphate; entactin; fibrin; fibronectin;glycolipids; glycophorin; glycoproteins; heparan sulphate; heparinsulphate; hyaluronic acid; immunglobulins; keratan sulphate; integrins;laminins; N-CAMs (Calcium independent Adhesive Molecules);proteoglycans; spektrin; vinculin; vitronectin.

Biopolymers:

Biopolymers are any biologically prepared molecule which, given theright conditions, can be assembled into polymeric, macromolecularstructures. Such molecules constitute important parts of theextracellular matrix where they participate in providing tissueresilience, strength, rigidity, integrity etc. Some importantbiopolymers with potential for use as bioactive coating on metalhydride-coated implants are: Alginates; Amelogenins; cellulose;chitosan; collagen; gelatins; oligosaccharides; pectin.

Blood Proteins:

This class of proteins typically contains any dissolved or aggregatedprotein which normally is present whole blood. Such proteins canparticipate in a wide range of biological processes like inflammation,homing of cells, clotting, cell signalling, defence, immune reactions,metabolism etc. Typical examples with potential for use as bioactivecoating on metal hydride-coated implants are: Albumin; albumen;cytokines; factor IX; factor V; factor VII; factor VIII; factor X;factor XI; factor XII; factor XIII; hemoglobins (with or without iron);immunoglobulins (antibodies); fibrin; platelet derived growth factors(PDGFs); plasminogen; thrombospondin; transferrin.

Enzymes:

Enzymes are any protein or peptide that have a specific catalytic effecton one ore more biological substrates which can be virtually anythingfrom simple sugars to complex macromolecules like DNA. Enzymes arepotentially useful for triggering biological responses in the tissue bydegradation of matrix molecules, or they could be used to activate orrelease other bioactive compounds in the implant coating. Some importantexamples with potential for use as bioactive coating on metalhydride-coated implants are: Abzymes (antibodies with enzymaticcapacity); adenylate cyclase; alkaline phosphatase; carboxylases;collagenases; cyclooxygenase; hydrolases; isomerases; ligases; lyases;metallo-matrix proteases (MMPs); nucleases; oxidoreductases; peptidases;peptide hydrolase; peptidyl transferase; phospholipase; proteases;sucrase-isomaltase; TIMPs; transferases.

Extracellular Matrix Proteins and Biomolecules:

Specialized cells, e.g., fibroblasts and osteoblasts, produce theextracellular matrix. This matrix participates in several importantprocesses. The matrix is crucial for i.e., wound healing, tissuehomeostasis, development and repair, tissue strength, and tissueintegrity. The matrix also decides the extracellular milieu like pH,ionic strength, osmolarity, etc. Furthermore, extracellular matrixmolecules are crucial for induction and control of biomineral formation(bone, cartilage, teeth). Important extracellular proteins andbiomolecules with potential for use as bioactive coating on metalhydride-coated implants include: Ameloblastin; amelin; amelogenins;collagens (I to XII); dentin-sialo-protein (DSP);dentin-sialo-phospho-protein (OSPP); elastins; enamelin; fibrins;fibronectins; keratins (1 to 2.0); laminins; tuftelin; carbohydrates;chondroitin sulphate; heparan sulphate; heparin sulphate; hyaluronicacid; lipids and fatty acids; lipopolysaccarides.

Growth Factors and Hormones:

Growth factors and hormones are molecules that bind to cellular surfacestructures (receptors) and generate a signal in the target cell to starta specific biological process. Examples of such processes are growth,programmed cell death, release of other molecules (e.g., extracellularmatrix molecules or sugar), cell differentiation and maturation,regulation of metabolic rate etc. Typical examples of such biomoleculeswith potential for use as bioactive coating on metal hydride-coatedimplants are: Activins (Act); Amphiregulin (AR); Angiopoietins (Ang 1 to4); Apo3 (a weak apoptosis inducer also known as TWEAK, DR3, WSL-I,TRAMP or LARD); Betacellulin (BTC); Basic Fibroblast Growth Factor(bFGF, FGF-b); Acidic Fibroblast Growth Factor (aFGF, FGF-a); 4-1BBLigand; Brain-derived Neurotrophic Factor (BDNF); Breast and Kidneyderived Bolokine (BRAK); Bone Morphogenic Proteins (BMPs); B-LymphocyteChemoattractant/B cell Attracting Chemokine 1 (BLC/BCA-1); CD27L (CD27ligand); CD30L (CD30 ligand); CD40L (CD40 ligand); AProliferation-inducing Ligand (APRIL); Cardiotrophin-1 (CT-1); CiliaryNeurotrophic Factor (CNTF); Connective Tissue Growth Factor (CTGF);Cytokines; 6-cysteine Chemokine (6Ckine); Epidermal Growth Factors(EGFs); Eotaxin (Eot); Epithelial Cell-derived Neutrophil ActivatingProtein 78 (ENA-78); Erythropoietin (Epo); Fibroblast Growth Factors(FGF 3 to 19); Fractalkine; Glial-derived Neurotrophic Factors (GDNFs);Glucocorticoid-induced TNF Receptor Ligand (GITRL); Granulocyte ColonyStimulating Factor (G-CSF); Granulocyte Macrophage Colony StimulatingFactor (GM-CSF); Granulocyte Chemotactic Proteins (GCPs); Growth Hormone(GH); I-309; Growth Related Oncogene (GRO); Inhibins (Inh);Interferon-inducible T-cell Alpha Chemoattractant (I-TAC); Fas Ligand(FasL); Heregulins (HRGs); Heparin-Binding Epidermal Growth Factor-LikeGrowth Factor (HB-EGF); fms-like Tyrosine Kinase 3 Ligand (Flt-3L);Hemofiltrate CC Chemokines (HCC-1 to 4); Hepatocyte Growth Factor (HGF);Insulin; Insulin-like Growth Factors (IGF 1 and 2); Interferon-gammaInducible Protein 10 (IP-10); Interleukins (IL 1 to 18);Interferon-gamma (IFN-gamma); Keratinocyte Growth Factor (KGF);Keratinocyte Growth Factor-2 (FGF-10); Leptin (OB); Leukemia InhibitoryFactor (LIF); Lymphotoxin Beta (LT-B); Lymphotactin (LTN);Macrophage-Colony Stimulating Factor (M-CSF); Macrophage-derivedChemokine (MDC); Macrophage Stimulating Protein (MSP); MacrophageInflammatory Proteins (MIPs); Midkine (MK); Monocyte ChemoattractantProteins (MCP-1 to 4); Monokine Induced by IFN-gamma (MIG); MSX 1; MSX2; Mullerian Inhibiting Substance (MIS); Myeloid Progenitor InhibitoryFactor 1 (MPIF-1); Nerve Growth Factor (NGF); Neurotrophins (NTs);Neutrophil Activating Peptide 2 (NAP-2); Oncostatin M (OSM);Osteocalcin; OP-1; Osteopontin; OX40 Ligand; Platelet derived GrowthFactors (PDGF aa, ab and bb); Platelet Factor 4 (PF4); Pleiotrophin(PTN); Pulmonary and Activation-regulated Chemokine (PARC); Regulated onActivation, Normal T-cell Expressed and Secreted (RANTES); Sensory andMotor Neuron-derived Factor (SMDF); Small Inducible Cytokine Subfamily AMember 26 (SCYA26); Stem Cell Factor (SCF); Stromal Cell Derived Factor1 (SDF-1); Thymus and Activation-regulated Chemokine (T ARC); ThymusExpressed Chemokine (TECK); TNF and ApoL-related Leukocyte-expressedLigand-1 (TALL-1); TNF-related Apoptosis Inducing Ligand (TRAIL);TNF-related Activation Induced Cytokine (TRANCE); Lymphotoxin InducibleExpression and Competes with HSV Glycoprotein D for HVEM T-Iymphocytereceptor (LIGHT); Placenta Growth Factor (PIGF); Thrombopoietin (Tpo);Transforming Growth Factors (TGF alpha, TGF beta 1, TGF beta 2); TumorNecrosis Factors (TNF alpha and beta); Vascular Endothelial GrowthFactors (VEGF-A, B, C and D); calcitonins; and steroid compounds such asnaturally occurring sex hormones such as estrogen, progesterone,testosterone as well as analogues thereof. Thus, certain implants, suchas IUD's (intrauterine devices) comprising e.g., estrogens orprogesterone or analogues thereof, could be contemplated.

Nucleic Acids (DNA):

DNA encodes the genes for proteins and peptides. Also, DNA contains awide array of sequences that regulate the expression of the containedgenes. Several types of DNA exist, depending on source, function,origin, and structure. Typical examples for DNA based molecules that canbe utilized as bioactive, slow release coatings on implants (localgene-therapy) are: A-DNA; B-DNA; artificial chromosomes carryingmammalian DNA (YACs); chromosomal DNA; circular DNA; cosmids carryingmammalian DNA; DNA; Double-stranded DNA (dsDNA); genomic DNA;hemi-methylated DNA; linear DNA; mammalian cDNA (complimentary DNA; DNAcopy of RNA); mammalian DNA; methylated DNA; mitochondrial DNA; phagescarrying mammalian DNA; phagemids carrying mammalian DNA; plasmidscarrying mammalian DNA; plastids carrying mammalian DNA; recombinantDNA; restriction fragments of mammalian DNA; retroposons carryingmammalian DNA; single-stranded DNA (ssDNA); transposons carryingmammalian DNA; T-DNA; viruses carrying mammalian DNA; Z-DNA.

Nucleic Acids (RNA):

RNA is a transcription of DNA-encoded information. (Sometimes (in someviruses) RNA is the essential information-encoding unit). Besides beingan intermediate for expression of genes, RNA have been shown to haveseveral biological functions. Ribozymes are simple RNA molecules with acatalytic action. These RNA can catalyze DNA and RNA cleavage andligation, hydrolyze peptides, and are the core of the translation of RNAinto peptides (the ribosome is a ribozyme). Typical examples of RNAmolecules with potential for use as bioactive coating on metalhydride-coated implants are: Acetylated transfer RNA (activated tRNA,charged tRNA); circular RNA; linear RNA; mammalian heterogeneous nuclearRNA (hnRNA), mammalian messenger RNA (mRNA); mammalian RNA; mammalianribosomal RNA (rRNA); mammalian transport RNA (tRNA); mRNA;poly-adenylated RNA; ribosomal RNA (rRNA); recombinant RNA; retroposonscarrying mammalian RNA; ribozymes; transport RNA (tRNA); virusescarrying mammalian RNA.

Receptors:

Receptors are cell surface biomolecules that bind signals (e.g., hormoneligands and growth factors) and transmit the signal Over the cellmembrane and into the internal machinery of cells. Different receptorsare differently “wired” imposing different intracellular responses evento the same ligand. This makes it possible for the cells to reactdifferentially to external signals by varying the pattern of receptorson their surface. Receptors typically bind their ligand in a reversiblemanner, making them suitable as carriers of growth factors that are tobe released into the tissue. Thus by coating implants with growth factorreceptors, and then load these receptors with their principal ligands, abioactive surface is achieved that can be used for controlled release ofgrowth factors to the surrounding tissues following implantation.Examples of suitable receptors with potential for use as bioactivecoating on metal hydride-coated implants includes: The CD class ofreceptors CD; EGF receptors; FGF receptors; Fibronectin receptor(VLA-5); Growth Factor receptor, IGF Binding Proteins (IGFBP 1 to 4);Integrins (including VLA 1-4); Laminin receptor; PDGF receptors;Transforming Growth Factor alpha and beta receptors; BMP receptors; Fas;Vascular Endothelial Growth Factor receptor (FLt-1); Vitronectinreceptor.

Synthetic Biomolecules

Synthetic biomolecules are molecules that are based on (mimicking)naturally occurring biomolecules. By synthesizing such molecules a widearray of chemical and structural modification can be introduced that canstabilize the molecule or make it more bioactive or specific. Thus if amolecule is either too unstable or unspecific to be used from extractsit is possible to engineer them and synthesize them for use as implantsurface coatings. Furthermore, many biomolecules are so low abundantthat extraction in industrial scales is impossible. Such rarebiomolecules have to be prepared synthetically, e.g., by recombinanttechnology or by (bio-) chemistry. Below is listed several classes ofsynthetic molecules that can be potentially useful for implant coatings:

Synthetic DNA:

A-DNA; antisense DNA; B-DNA; complimentary DNA (cDNA); chemicallymodified DNA; chemically stabilized DNA; DNA; DNA analogues; DNAoligomers; DNA polymers; DNA-RNA hybrids; double-stranded DNA (dsDNA);hemi-methylated DNA; methylated DNA; single-stranded DNA (ssDNA);recombinant DNA; triplex DNA; T-DNA; Z-DNA.

Synthetic RNA:

Antisense RNA; chemically modified RNA; chemically stabilized RNA;heterogeneous nuclear RNA (hnRNA); messenger RNA (mRNA); ribozymes; RNA;RNA analogues; RNA-DNA hybrids; RNA oligomers; RNA polymers; ribosomalRNA (rRNA); transport RNA (tRNA).

Synthetic Biopolymers:

Cationic and anionic liposomes; cellulose acetate; hyaluronic acid;polylactic acid; polyglycol alginate; polyglycolic acid; poly-prolines;polysaccharides.

Synthetic Peptides:

Decapeptides containing DOPA and/or diDOP A; peptides with sequence “AlaLys Pro Ser Tyr Pro Pro Thr Tyr Lys”; peptides where Pro is substitutedwith hydroxyproline; peptides where one or more Pro is substituted withDOPA; peptides where one or more Pro is substituted with diDOP A;peptides where one or more Tyr is substituted with DOPA; peptidehormones; peptide sequences based on the above listed extractedproteins; peptides containing an RGD (Arg Gly Asp) motif.

Recombinant Proteins:

All recombinantly prepared peptides and proteins.

Synthetic Enzyme Inhibitors:

Synthetic enzyme inhibitors range from simple molecules, like certainmetal ions, that block enzyme activity by binding directly to theenzyme, to synthetic molecules that mimic the natural substrate of anenzyme and thus compete with the principle substrate. An implant coatingincluding enzyme inhibitors could help stabilizing and counteractbreakdown of other biomolecules present in the coating, so that morereaction time and/or higher concentration of the bioactive compound isachieved. Examples of enzyme inhibitors are: Pepstatin; poly-pro lines;D-sugars; D-aminoacids; Cyanide; Diisopropyl fluorophosphates (DFP);metal ions; N-tosyl-I-phenylalaninechloromethyl ketone (TPCK);Physostigmine; Parathion; Penicillin.

Vitamins (Synthetic or Extracted) for Incorporation in Hydride:

Biotin; calciferol (Vitamin D's; vital for bone mineralisation); citrin;folic acid; niacin; nicotinamide; nicotinamide adenine dinucleotide(NAD, NAD+); nicotinamide adenine dinucleotide phosphate (NADP, NADPH);retinoic acid (vitamin A); riboflavin; vitamin Bs; vitamin C (vital forcollagen synthesis); vitamin E; vitamin Ks.

Other Bioactive Molecules for Incorporation into Hydride:

Adenosine di-phosphate (ADP); adenosine mono-phosphate (AMP); adenosinetri-phosphate (ATP); amino acids; cyclic AMP (cAMP);3,4-dihydroxyphenylalanine (DOPA); 5′-di(dihydroxyphenyl-L-alanine(diDOPA); diDOPA quinone; DOPA-like o-diphenols; fatty acids; glucose;hydroxyproline; nucleosides; nucleotides (RNA and DNA bases);prostaglandin; sugars; sphingosine 1-phosphate; rapamycin; synthetic sexhormones such as estrogen, progesterone or testosterone analogues, e.g.,Tamoxifene; estrogen receptor modulators (SERMs) such as Raloxifene;bis-phosphonates such as alendronate, risendronate and etidronate;statins such as cerivastatin, lovastatin, simvaststin, pravastatin,fluvastatin, atorvastatin and sodium3,5-dihydroxy-7-[3-(4-fluorophenyl)-1-(methylethyl)-1H-indol-2-yl]-hept-6-enoate.

Drugs for Incorporation into Hydride Coatings:

Drugs incorporated in the hydride layer could be utilized for localeffects like improving local resistance against invading microbes, localpain control, local inhibition of prostaglandin synthesis; localinflammation regulation, local induction of biomineralisation and localstimulation of tissue growth. Examples of drugs suitable forincorporation into metal hydride layers include: Antibiotics;cyclooxygenase inhibitors; hormones; inflammation inhibitors; NSAIDs;painkillers; prostaglandin synthesis inhibitors; steroids, tetracycline(also as biomineralizing agent).

Biologically Active Ions for Incorporation in Hydride Coatings:

Ions are important in a diversity of biological mechanisms. Byincorporating biologically active ions in metal hydride layers onimplants it is possible to locally stimulate biological processes likeenzyme function, enzyme blocking, cellular uptake of biomolecules,homing of specific cells, biomineralization, apoptosis, cellularsecretion of biomolecules, cellular metabolism and cellular defense.Examples of bioactive ions for incorporation into metal hydride include:Calcium; chromium; copper; fluoride; gold; iodide; iron; potassium;magnesium; manganese; selenium; silver; sodium; zinc.

Marker Biomolecules:

Biological Markers are molecules that generates a detectable signal,e.g., by emitting light, enzymatic activity, radioactivity, specificcolor, magnetism, x-ray density, specific structure, antigenicity, etc.,that can be detected by specific instruments or by microscopy or animaging method like x-ray or magnetic resonance. Markers are used tomonitor biological processes in research and development of newbiomedical treatment strategies. On implants, such markers wouldtypically be employed to monitor processes like biocompatibility,formation of tissue, tissue neogenesis, biomineralisation, inflammation,infection, regeneration, repair, tissue homeostasis, tissue breakdown,tissue turnover, release of biomolecules from the implant surface,bioactivity of released biomolecules, uptake and expression of nucleicacids released from the implant surface, and antibiotic capability ofthe implant surface to provide “proof of principle”, effect, efficacyand safety validation prior to clinical studies.

Marker biomolecules suitable for incorporation in hydride coatingsinclude: Calcein; alizaran red; tetracyclins; fluorescins; fura;luciferase; alkaline phosphatase; radioed amino acids (e.g., marked with³²P, ³³P, ³H, ³⁵S, ¹⁴C, ¹²⁵I, ⁵¹Cr, ⁴⁵CaO; radiolabeled nucleotides(e.g., marked with ³²p, ³³p, ³H, ³⁵S, ¹⁴C,); radiolabeled peptides andproteins; radio labeled DNA and RNA; immuno-gold complexes (goldparticles with antibodies attached); immuno-silver complexes;immuno-magnetite complexes; Green Fluorescent protein (GFP); RedFluorescent Protein (E5); biotinylated proteins and peptides;biotinylated nucleic acids; biotinylated antibodies; biotinylatedcarbon-linkers; reporter genes (any gene that generates a signal whenexpressed); propidium iodide; diamidino yellow.

The device or implant according to the invention can be used for anumber of purposes. Examples of such purposes include use for: inducinglocal hard tissue (e.g., bone tissue) formation at the implantationsite; controlling microbial growth and/or invasion at the implantationsite or systemically; reducing inflammation at the implantation site orsystemically; stimulating ligament repair, regeneration or formation;inducing cartilage formation; nucleating, controlling and/or templatingbiomineralization; improving attachment between implants and tissues;improving osseointegration of implants; improving tissue adherence to animplant; hindering tissue adherence to an (semipermanent or temporary)implant; improving contact between tissues or tissues and implants,improving tissue sealing of a (surgical) wound; inducing apoptosis (celldeath) in unwanted cells (e.g., cancer cells); inducing specific celldifferentiation and/or maturation, increasing tissue tensile strength;improving wound healing; speeding up wound healing; templating tissueformation; guiding tissue formation; local gene therapy; stimulatingnerve growth; improving vascularisation in tissues adjacent to animplant; stimulating local extracellular matrix synthesis; inhibitinglocal extracellular matrix breakdown; inducing local growth factorrelease; increasing local tissue metabolism; improving function of atissue or body-part; reducing local pain and discomfort. The purposewill depend on the type of implant as well as the nature and/orconcentration of the biomolecule present in the hydride layer on theimplant.

When the metal material (A) is an alloy of titanium, zirconium,tantalum, hafnium or niobium, it may be an alloy between one or more ofthese metal elements; or it may be an alloy containing one or more othermetals such as aluminium, vanadium, chrome, cobalt, magnesium, iron,gold, silver, copper, mercury, tin or zinc; or both.

It is preferred that the metal material (A) is titanium or an alloythereof, e.g., an alloy with zirconium, tantalum, hafnium, niobium,aluminium, vanadium, chrome, cobalt, magnesium, iron, gold, silver,copper, mercury, tin or zinc. In a particularly preferred embodiment,the metal material (A) is titanium.

The corresponding hydride material (B) is preferably titanium hydride.

The amount of biomolecule substance (C) present on or in the hydridelayer (B) of the parts of the prosthesis, device or implant coated withthe hydride may vary within wide limits, e.g., dependent on the chemicaland biological characteristics of the biomolecule substance orsubstances in question. Thus, the biomolecule substance (C) associatedwith the hydride material (B) may be present in amounts ranging from aslow from 1 picogram per mm to as high as 1 mg per mm² of hydride-coateddevice or implant surface. However, it is contemplated that most usefulbiomolecule coatings will range from 0.1 nanogram to 100 microgram permm².

As indicated above, the method of the invention involves subjectingsurface parts of the metal material (A) to a electrolysis treatment toform the hydride layer (B), said treatment being carried out in thepresence of one or more biomolecule substances as discussed above. Ithas been found that is important that the conditions in the electrolyte(pH, ionic strength etc.) are such that the biomolecule has a netpositive charge. It is therefore advantageous that most biomolecules areampholytes, i.e., they are weak acids (or bases) that change their netcharge according to the ionic strength and pH of the solution they aredissolved in. Consequently, the main concern for incorporation thereofin a hydride layer is stability under the conditions needed forbio-hydride preparation, i.e., an environment that supply enough H+ ionsfor hydride preparation and at the same time keeps the net charge of thebiomolecule in question positive. This mostly means that the electrolyteshould have a high salt concentration and hence ionic strength; acomparatively high temperature, although preferably below any denaturingtemperature of the biomolecule substance; and a low pH.

Thus, the electrolyte may be any salt solution, preferably aqueous,e.g., a solution of sodium chloride, sodium sulphate, calcium phosphate,calcium chloride, phosphate buffered saline (PBS), saline, a saltsolution mimicking physiological conditions, bicarbonates, carbonatesetc., in which the desired biomolecule is dissolved. The ionic strengthof the salt is typically 1M, but concentrations can be adjusted to aslow as 0.01 M and as high as 10M according to the chemical propertiesand concentration of the biomolecule(s).

The temperature of the electrolyte containing the biomolecule may rangefrom ambient (20° C.) to as high as the boiling point of theelectrolyte, typically around 100° C., although the use of temperaturesin the upper part of this range clearly depends on the ability of thebiomolecule to withstand such temperatures without damage. If thebiomolecule can withstand it, an optimum temperature for the formationof hydride is around 80° C.

The pH of the electrolyte is typically adjusted to the desired pH bymeans of a strong acid, e.g., HCl, HF, H₂SO₄, etc., although it shouldbe taken into account that a pH below 2 will produce a irregular,corroded implant surface on titanium while a pH above 2 conserves theoriginal surface. The pH is adjusted according to the desiredHydridelbiomolecule ratio; Low pH produces an implant surface with ahigh hydridelbiomolecule ratio (=more metal hydride), whereas a high pHclose to the pI of the biomolecule in question will produce a surfacewith a low hydridelbiomolecule ratio (=more biomolecules). Accordingly,while any pH between 0 and 10 can be used, the preferred pH for hydridepreparation is between 5 and 2, depending on the chemicalcharacteristics and concentration of the biomolecule(s), the electrolyteused and the preferred hydridelbiomolecule ratio. For higherhydridelbiomolecule(s) ratios (=more hydride), adjust pH more acidic,for lower hydridelbiomolecule(s) ratios (=more biomolecule(s)) adjust pHcloser to, but not above, Pi_(BIOMOLECULE). The only requirement is thatthere are hydrogen ions (H⁺) and positively charged biomolecules(Biomolecule+, net charge) present in the electrolyte.

The concentration of the biomolecule(s) (one or any combinations of twoor more) in the electrolyte may vary over a very wide range, dependingon type of bioactivity, type of molecule, chemical and biologicalcharacteristics, toxicity, potency, mode of action, if it is to bereleased or not from the hydride layer, stability in vivo, stability inthe electrolyte, availability, optimal pH, etc., Thus, the concentrationof the biomolecule(s) in the electrolyte may be within the range of 1 pgto 50 mg per milliliter. A preferred range is between 10 pg and 1 mg permilliliter, but the optimal biomolecule concentration should always beby finally determined in pilot experiments with each biomolecule orbiomolecule-mix. Also, the time span over which the electrolysis isperformed may vary, but chiefly influences the thickness of the hydridelayer and hence the concentration of biomolecules in the hydride layer.

An electrolysis cell for use in the method of the invention may be ofany conventional design, but is typically a two-chamber cell without anyconducting connections between the chambers except for the electrolyte.The metal implant to be hydride-modified is placed in the cathode (i.e.,the negatively charged electrode) chamber, whereas the anode (thepositively charged electrode), typically made of carbon, is placed in aseparate chamber. The electrolytes of each chamber are connected througha porous glass or porcelain filter allowing the current to passunhindered, but without any exchange of electrolytes between the twochambers. This is important because the products from the anodereaction, e.g., chloride or hypo-chlorites etc., could potentiallyinterfere with the formation of the biomolecule-hydride layer or destroyor modify the biomolecule in the cathode electrolyte. The separation ofthe two cells also allows the use of a smaller cathode electrolytevolume and thus a more effective use of biomolecules as well as thepossibility to use a two-electrolyte system that allows optimization ofthe electrolytic process, e.g., one electrolyte optimal for biomoleculeson the cathode side and an electrolyte on the anode side which isoptimized for the efficacy of the electrolysis per se (conductivity,avoiding toxic products, or even producing useful byproducts/coatings).

As indicated above, the temperature in the cathode cell (T_(cat)) shouldbe as high at possible with an optimum for hydride preparation at 80° C.

The electrolytic process itself also produces heat which can pose twoproblems: constituents of the electrolyte will evaporate so that thevolume decreases and the ionic strength and the concentration ofbiomolecules increase above the preferred range, and the increase intemperature might cause precipitation, coagulation, denaturation,degradation or destruction of the biomolecule(s) present. Therefore, thecathode compartment of the electrolysis cell is preferably equipped witha cooled lid for condensation of vaporized electrolyte and a temperatureregulated radiator shell for stabilizing temperatures and volumes duringelectrolysis.

By adjusting current, charge and electrolyte composition it may also bepossible to provide a favorable milieu for positive charge for mostbiomolecules. If not, a pulse field electrolysis set-up where thepolarity of the electrodes is switching in controlled cycles duringpreparation of the bio-hydride layer could be one way to omit a negativenet charge problem.

The power supply is typically a so-called current pump, i.e., a devicedelivering a constant current even if the resistance within the circuitvaries. Although voltages between 0.1 and 1000 volts can be used, thevoltage is typically below 10 volts. The current density duringelectrolysis is typically in the range of 0.1 mA to 1 A per squarecentimeter (cm²) of implant specimen. A preferred charge density is IMa/cm², although adjustments in the electrolyte, pH and temperature toincrease biomolecule compatibility may command minor or major deviationsfrom this value.

The duration of the process depends on several parameters, such as thedesired thickness of the bio-hydride layer, the composition andcharacteristics of the electrolyte, the characteristics of thebiomolecule, the temperature and pH, the desired hydridelbiomoleculeratio, the size of the implant specimen, the volume of the cathodeelectrolyte, the concentration of the biomolecule, etc. Thus, theduration of the process may be between 0.5 hours and several days.However, an optimal time-span is generally between 8 and 24 hours.

To monitor the bio-hydride process, a calomel electrode may typically beplaced in the cathode chamber. When the hydride layer formation processat the cathode is optimal, a difference of −1 Volt is observed betweenthe calomel electrode and the cathode. If the current differs much fromthis value, the process will be running under sub-optimal conditions,and a change in the set-up should be considered. Furthermore, atemperature probe and a pH probe may typically be placed in the cathodechamber to monitor that the process is running within the desired pH andtemperature limits. A stirring device, such as a magnetic stirrer, mayalso be applied in the cathode cell to continuously mix the electrolyteand keep the temperature homogenous and avoid variations in local ionicstrength, pH and biomolecule concentrations.

After the electrolysis step, the now biomolecule/hydride-coated metaldevice or implant is immediately removed from the electrolyte andtreated according to the requirement of the biomolecule(s) in question.Typically, the device or implant specimen is allowed to air-dry and isthen packaged in a sterile, airtight plastic bag in which it is storeduntil use for implantation. However, some biomolecules might besensitive to drying, and consequently a wet storage system might bedesired, e.g., like canning or storage in a fluid like saline or simplythe electrolyte from the manufacturing process. Although theelectrolysis can be run under aseptic or even sterile conditions, theneed for doing this may be avoided by including a sterilization stepprior to use, using conventional methods, such as ionizing radiation,heating, autoclaving, or ethylene oxide gas, etc. The choice of methodwill depend on the specific characteristics and properties of thebiomolecule(s) present in the metal hydride layer.

Prior to the electrolysis treatment, the device or implant should bethoroughly cleaned. This may typically consist in the implant beingmechanically pre-treated by electropolishing or sandblasting to modifysurface structure if desired, and subsequently thoroughly cleaned usinghot caustic soda followed by a de-greasing step, e.g., in concentratedtri-chloro-ethylene, ethanol or methanol, before being treated in apickling solution, e.g., hydrofluoric acid, to remove oxides andimpurities on the surface. After pickling, the implant specimen iswashed thoroughly in hot, double distilled, ion-exchanged water.

The invention is further illustrated by the following, non-limitingexamples of which Examples 1-4 describe conducted experiments, andExamples 5-11 illustrate contemplated working examples.

EXAMPLES Example 1

Preparation of a Titanium Hydride Implant Surface Layer Containing anExtracellular Matrix Protein.

A two-chamber electrolysis cell was used to prepare a layer of titaniumhydride containing the extracellular matrix molecule amelogenin ontofive coin-shaped electropolished titanium implants each with a surfacearea of 0.6 cm² exposed to the electrolyte. Five similar items were usedas controls by being present in the electrolyte chamber, but notconnected to the electrolysis current. The electrolyte in both chamberswas 1M NaCl in sterile water, pH adjusted to pH 4 by the use of HCl, andthe initial concentration of amelogenin was 0.1 mg/ml. For electrolysisa voltage of 10 volts at a charge density of 1 mA/cm² was used. Thetemperature of the cathode chamber was set to 70° C. Electrolysis wasallowed to progress for 18 hours, after which the titanium implants wereremoved from the electrolysis cell, washed in sterile water and allowedto air-dry in a desiccator.

After drying the titanium test and control specimens were each washedthree times in 1 ml saline at pH 6.5. Following the washes, any proteinremaining on the titanium surfaces was dissolved by boiling the titaniumspecimen in 0.5 ml 2×SDS-PAGE sample buffer (0.4 g SDS, 1.0 g2-mercaptoethanol, 0.02 g bromophenol blue and 4.4 g glycerol in 10 ml0.125 M Tris/HCl, pH 6.8). The washing solutions and the 2×SDS-PAGEsample buffer with possible protein therein were precipitated with anequal volume of 0.6 N perchloric acid and the supernatant was cleared bycentrifugation. The precipitation pellets, containing salt and possibleorganic molecules, were then dissolved in 50 μl 2×SDS-PAGE sample bufferand boiled for five minutes. All samples were then submitted toelectrophoresis on a 12% SDS-polyacrylamide gel at 80 mA overnight.After electrophoresis, proteins in the gel were transferred onto apoly(vinylidene difluoride) membrane by the semidry “sandwich”electroblotting technique. Amelogenin proteins were then detected by animmune assay using an rabbit amelogenin specific primary IgG antibodyand a biotin labelled goat anti rabbit IgG secondary antibody. Thewestern blot showed significant amounts of amelogenins present inextracts from test specimens, and hence trapped in the titanium hydridelayer thereon, whereas no amelogenins were detected in extracts from thecontrol specimens that were not connected to the electrolysis current.

This experiment clearly demonstrates that a significant amount ofamelogenin was incorporated in the hydride layer during the electrolyticprocess. The amelogenin proteins were not merely present as a simplecoating, since there is no evidence of proteins in the initial washingsolutions. Only with the combination of a strong detergent (SDS), areducing agent (mercaptoethanol) and high temperature (100° C.) couldamelogenins be extracted from the titanium hydride surface layer anddetected by western blot. The amount of protein extracted was calculatedto be 50 μg/cm² by comparison with an amelogenin standard. This figureis well within the bioactivity range of this extracellular matrixprotein.

Example 2

Production of An Amelogenin-containing Titanium Hydride Implant SurfaceLayer.

The set-up from example one was used to produce a layer of titaniumhydride containing the extracellular matrix molecule amelogenin ontoelectropolished titanium implants with a surface area of 0.35 cm²exposed to the electrolyte. The electrolyte in both chambers was 1M NaClin sterile water, pH adjusted to pH 4 by the use of HCl, and the initialconcentration of amelogenin was 0.1 mg/ml. For electrolysis a voltage of10 volts at a charge density of 1 mA/cm² was used. Tcat was set to 70°C. Electrolysis was allowed to progress for 18 hours after which thetitanium implants were removed from the electrolysis cell, washed insterile water and allowed to air-dry in a desiccator.

After drying, the titanium specimens were washed three times in 1 mlsaline at pH 6.5. Following the washes the proteins remaining on thetitanium surfaces were dissolved by boiling the titanium specimen in 0.1ml 2×SDS sample buffer (0.4 g SDS, 1.0 g 2-mercaptoethanol in 10 ml0.125 M Tris/HCl, pH 6.8) for 5 minutes. The amount of amelogenindissolved into the SDS solution from the rinsed titanium surfaces wasthen analyzed by standard photometry measuring light absorbance at 280and 310 nm against a 2×SDS sample buffer blank, and comparing theresults with a standard dilution series of amelogenin in 2×SDS samplebuffer. The experiment was repeated twice in series of 16 implants, bothtimes with 5 negative internal controls in the form of identicaltitanium implants that was present in the reaction chamber during thewhole process, but not attached to the cathode.

This experiment clearly demonstrates that a significant amount ofamelogenin was incorporated in the hydride layer during the electrolyticprocess. The amelogenin proteins were not only present as a simplecoating, as there is no evidence of proteins in the washing solutions.Only with the combination of a strong detergent (SDS), a reducing agent(mercaptoethanol) and high temperature (100° C.) could amelogenins beextracted from the surface layer of the titanium hydride. the amount ofprotein extracted was calculated to range between 57 and 114 μg/cm² witha mean value of 87 μg amelogenin per cm², by comparison with theamelogenin standard. This figure is well within the bioactivity range ofthis extracellular matrix protein. Identical control implants that hadbeen present is the same electrolytic cell as the experimental implants,but which were not connected to the cathode, showed no significantamounts of amelogenin proteins attached to the surface (<1 μg/cm²).

Example 3

Production of a Nucleic Acid-containing Titanium Hydride Implant SurfaceLayer.

The set-up from example one was used to produce a layer of titaniumhydride containing nucleic acids in the form of radio labeled totalhuman placenta DNA onto electropolished titanium implants with a totalsurface area of 0.35 cm² exposed to the electrolyte. The electrolyte inboth chambers was 1 M NaCl in sterile water. The pH was adjusted to pH 2by the use of HCL The initial concentration of DNA in the electrolytewas 10 μg/ml. For electrolysis a voltage of 10 volts at a charge densityof 1 mA/cm and a T_(cat) of 75° C. were used. Electrolysis was allowedto progress for 16 or 24 hours after which the titanium specimens wereremoved from the electrolysis cell, rinsed three times in ample amountsof Tris-EDTA buffer (TE-buffer; 10 mM Tris-Cl and 1 mM EDTA in sterilewater, pH 7.6) and then allowed to air dry over night in a desiccator.

The DNA was radiolabeled using a Stratagene Prime-It® II Random PrimerLabeling kit for production of high specific-activity probes and[α-³²]dATP (Amersham). After labeling of the DNA, the specificradioactivity of the DNA probe was measured in a Packard Tricarb®scintillation counter to be 3.0×10⁸ disintegrations per minute permicrogram labeled DNA (dpm/μg).

After drying the titanium specimens with tentative nucleic acidsattached, were put on a phosphor screen (Fujii®) for 15 minutes. Thespecimens were then removed and the phosphor screen was scanned in aBioRad® phosphor imaging machine measuring the number of disintegrationsoccurred at the surface of each implant using a 100/μm grid (12265points per implant) The experiment was repeated twice in a series of 16implants, both times with 5 negative internal controls in the form ofidentical titanium implants that was present in the reaction chamberduring the whole process, but which were not connected to the cathode.For the first series the reaction time was 24 hours, for the second itwas 16 hours. The total number of dpm per implant was calculated andconverted to μg DNA per square centimeter (μg DNA/cm²).

The amount of DNA present on the implants ranged between 0.25 and 0.75μg/cm² with a mean value of 0.43 μg DNA per cm² when the reaction timewas 24 hours. When the reaction time was reduced to 16 hours, therespective values ranged between 0.19 and 0.32 μg/cm² with a mean valueof 0.30 μg DNA per cm². This figure is well within the applicable rangefor gene therapy and DNA vaccines and other molecular medicineapplications. Identical control implants that had been present is thesame electrolytic cell as the experimental implants, but that were notconnected to the cathode showed only very small amounts (picograms) ofDNA attached to the surface.

This experiment clearly demonstrates that a significant amount of DNAwas incorporated in the hydride layer during the electrolytic process.The DNA was not merely present as a simple coating because the DNA wasnot dissolved or washed off the test implants during rinsing with TE.Furthermore, the fact that the amount of DNA incorporated in thetitanium hydride surface layer increased linearly with reaction timealso shows that adjusting reaction time is an easy way to control theamount of biomolecules in the hydride layer.

Example 4

Preparation of a Titanium Hydride Implant Surface Layer ContainingAscorbic Acid

The set-up from Example 1 was used to prepare a layer of titaniumhydride containing ascorbic acid (vitamin C) onto electropolishedcoin-shaped titanium implants with a total surface area exposed to theelectrolyte of 0.35 cm². The electrolyte in both chambers was salinewith pH adjusted to pH 3 by means of phosphoric acid. The initialconcentration of ascorbic acid was 10 mg/ml. Electrolysis with a voltageof 6 volts at a Current density of 2 mA/cm² and a cathode chambertemperature of 20° C. was used. Electrolysis was allowed to progress for16 hours after which the titanium implant is removed from theelectrolysis cell, rinsed twice in sterile water and allowed to dry in adesiccator.

After drying over night, the tentative ascorbic acid was dissolved fromthe titanium specimens by submerging the specimens in 1 ml Tris-EDT Abuffer (TE-buffer; 10 mM Tris-Cl and 1 mM EDT A in sterile water) at pH8.0 for 1 hour with shaking. The amount of ascorbic acid in the buffersamples was then analyzed by measuring light absorption at 250 nm andcomparing the results with a standard curve for ascorbic acid in TE, pH8.0 at this wavelength. Identical control implants present is the sameelectrolytic cell as the experimental implants, but not connected to thecathode may be used as controls. The experiment was repeated twice in aseries of 16 implants, both times with 5 negative, internal controls.

The amount of ascorbic acid extracted from the titanium specimens wascalculated to range between 28 and 76 μg/cm² with a mean value of 39 μgascorbic acid per cm², by comparison with the ascorbic acid standard.This figure is well within the bioactivity range of this vitamin (thenormal plasma concentration in humans range between 8-15 μg/ml). Theinternal control specimens that had been present is the sameelectrolytic cell as the experimental implants, but which were notconnected to the cathode, showed only minute amounts of ascorbic acidattached to the surface (4 μg/cm²). This experiment clearly demonstratesthat a biologically significant amount of ascorbic acid can beincorporated or attached to the titanium hydride layer during theelectrolytic process.

Example 5

Preparation of a Titanium Hydride Implant Surface Layer Containing aSynthetic Growth Factor-based Peptide

The set-up from Example 1 may be used to prepare a layer of titaniumhydride containing a synthetic, full-length (37 amino acids) fibroblastgrowth factor 4 (FGF-4) peptide onto coin-shaped electropolishedtitanium implants with a total surface area of 0.6 cm² exposed to theelectrolyte. Electrolytes, pH, voltage, current density and electrolysistime may suitably be as in Example 1. The initial concentration of FGF-4may suitably be 0.1 mg/ml, and the cathode chamber temperature maysuitably be 50° C.

Following washing in saline and 2×SDS-PAGE buffer, precipitation,centrifugation, re-dissolution in SDS-PAGE, boiling and electrophoresisas in Example 1, protein in the gel may be transferred to a silverstaining solution and the full-length synthetic FGF-4 peptides presentvisualised as a distinct band in the gel. Identical control implantsincorporated in the same electrolytic cell as the experimental implants,but not connected to the cathode, can be used as controls.

Example 6

Preparation of a Titanium Hydride Implant Surface Layer Containing anAntibiotic

The set-up from Example 1 may be used to prepare a layer of titaniumhydride containing the antibiotic agent amoxicillin (aminopenicillinium)onto an electropolished, coin-shaped titanium implant with a surfacearea exposed to the electrolyte of 0.6 cm². The electrolyte in bothchambers is suitably 1M NaCl in sterile water with pH adjusted to pH 2by means of HCl, and the initial concentration of amoxicillin issuitably 5 mg/ml. For electrolysis a voltage of 10 volts at a chargedensity of I mA/cm² and a cathode chamber temperature of 50° C. may beused. Electrolysis may suitably be allowed to progress for 24 hoursafter which the titanium implant is removed from the electrolysis cell,rinsed in sterile water and allowed to dry in a desiccator.

After drying the amount of amoxicillin trapped in the hydride layer onthe titanium implants may be assessed by its antibacterial effect onpenicillin sensitive bacteria of the species Escherichia coli (E. coli),strain K12, in liquid cultures. The cultures are suitably inoculatedwith one colony of E. coli K12 in 5 ml LB broth. After inoculation, themodified implants and controls are placed in the culture and thecultures incubated at 37° C. overnight. The next day the amounts ofbacteria present in the cultures may be assessed by photometry andcomparison with a standard dilution. Identical control implants presentin the same electrolytic cell as the experimental implants, but notconnected to the cathode may be used as controls.

Example 7

Preparation of a Biomineral-inducing Titanium Hydride Implant SurfaceLayer

The set-up from Example 1 may be used to prepare a layer of titaniumhydride containing a synthetic poly-proline peptide that has thepotential to act as a biological nucleator of mineral formation insaturated solutions of calcium phosphate. The biomolecule may beincorporated in the hydride layer on electropolished, coin-shapedtitanium implants surface with a total area exposed to the electrolyteof 0.6 cm². The electrolyte in both chambers may suitably be 1M NaCl insterile water with pH adjusted to pH 2 by means of HCl, and the initialconcentration of the synthetic poly-proline may suitably be 0.1 mg/ml.For electrolysis a voltage of 10 volts at a current density of 1 mA/cm²and a cathode chamber temperature of 70° C. may be used. Electrolysismay suitably be allowed to progress for 18 hours after which thetitanium implants are removed from the electrolysis cell, rinsed insterile water and allowed to air-dry in a desiccator.

After drying the titanium implants and controls with tentative mineralnucleating peptide attached are placed in 5 ml saturated solution ofcalcium phosphate. After incubation for 4 hours in room temperature, theimplants are removed from the mineral solution, rinsed in sterile waterand air-dried in a desiccator. When dry, the implants may be directlysubmitted to scanning electron microscopy for assessment of the numberof mineral foci present on the modified surfaces. Identical controlimplants present is the same electrolytic cell as the experimentalimplants but not connected to the cathode may be used as controls.

Example 8

Preparation of a Swelling (Space Filling) Biomolecule-titanium-hydrideImplant Surface Layer.

The set-up from Example 1 may be used to prepare a layer of titaniumhydride containing Ca-alginate nanospheres (Pronova AS) ontoelectropolished, coin-shaped titanium implants with a total area exposedto the electrolyte of 0.6 cm². The electrolyte in both chambers issuitably 1M CaCl₂ in sterile water with pH adjusted to pH 5.5 by meansof HCl, and the initial concentration of Ca-alginate is suitably 1% w/v.For electrolysis a voltage of 10 volts at a current density of 1 mA/cm²and a cathode chamber temperature of 35° C. may be used. Electrolysis issuitably allowed to progress for 48 hours, after which the titaniumimplants are removed from the electrolysis cell, rinsed in cold sterilewater and allowed to air-dry in a desiccator.

After drying, the titanium implants with a hydride-alginate layer aresuitably submerged in sterile saline, dyed with bromophenol blue (0.02g/ml) and incubated for one hour at 37° C. with the modified surfacefacing the solution. Following incubation in the dyed saline theimplants and controls are rinsed in distilled water and observed with amagnifying glass for the retention of blue dye within the tentativeswelled alginate layer. The thickness of the alginate layers may also beassessed by viewing the implants edge on in a calibrated lightmicroscope. Identical control implants present is the same electrolyticcell as the experimental implants but not connected to the cathode maybe used as controls.

Example 9

Preparation of a Dual Layer Biomolecule-titanium-hydride Implant Surface

The set-up from Example 1 may be used to prepare a dual layer ofbiomolecule containing titanium hydride on the surface ofelectropolished, coin-shaped titanium implants with a total surfaceexposed to the electrolyte of 0.6 cm². The inner layer may be preparedusing amelogenin as biomolecule according to the method in Example 1.Immediately after this procedure, and without air-drying in between, theelectrolyte and conditions may be changed to those of Example 3 usinggenomic human DNA as biomolecule. In this way titanium implants may beprepared with an outer layer of titanium hydride-DNA overlaying an innerlayer of titanium hydride-amelogenin. After the electrolysis theimplants are removed from the electrolysis cell, rinsed in sterile waterand allowed to air-dry in a desiccator.

After drying the titanium specimens with tentative nucleic acids andproteins attached are suitably rinsed three times in Tris-EDTA buffer(TE-buffer; 10 mM Tris-Cl and 1 mM EDT A in sterile water). At eachrinse the pH is increased starting at pH 7.4, then rinsed at pH 7.6 andfinally at pH 8.0. After rinsing in TE the remaining DNA and protein onthe titanium implants is finally removed using 0.1 N NaOH. The rinsingfractions are then divided in two; on part for nucleic acid analysis andone for protein analysis. The DNA fractions are suitably precipitatedwith an equal volume of absolute alcohol at −20° C. for 1 hour and thencleared from the supernatant by centrifugation at 13,000 g at 4° C. Thepellet is then dissolved in 50 μl TE buffer pH 7.4 and the amount of DNAfrom all four rinsing solutions assessed by fluorometric analysis usingHoechst dye (Boehringer Mannheim).

The fractions for protein analysis are suitably precipitated with anequal volume of 0.6 N perchloric acid and the supernatants cleared bycentrifugation. The precipitation pellets containing salt and proteinsare then dissolved in 50 μl 2×SDS-PAGE sample buffer (0.4 g SDS, 1.0 g2-mercaptoethanol, 0.02 g bromophenol blue and 4.4 g glycerol in 10 ml0.125 M Tris/HCl, pH 6.8) and boiled for five minutes. All samples arethen submitted to electrophoresis on a 10% SDS-polyacrylamide gel at 80mA for 4 hours. After electrophoresis proteins in the gel aretransferred to a silver staining solution and amelogenin present in thefractions is visualized as distinct bands in the gel. Identical controlimplants present is the same electrolytic cell as the experimentalimplants but not connected to the cathode may be used as controls.

Example 10

Preparation of a Dual Zone Biomolecule-titanium-hydride Layered ImplantSurface.

The set-up from Example 1 may be used to prepare two separate zones oftitanium hydride layers. Electropolished, rod-shaped titanium implantswith a total area of 2 cm² were treated according to Examples 3 and 6.First the implants were placed in the electrolyte from Example 3, sothat only one half of each implant was submerged in the electrolyte.After the procedure of Example 3 was completed, the implants were turnedaround and placed in a new electrolyte similar to the one used inExample 6, so that the untreated half of each implant now was submergedin electrolyte. The procedure and reaction conditions from Example 6were then carried out, after which the titanium specimen was removedfrom the electrolysis cell, rinsed in sterile water and allowed to dryin a desiccator.

Following electrolysis the dual zone implants are cut in two at thecenter. The halves layered with titanium hydride-synthetic FGF-4 peptidemay be submitted to analysis according to Example 2. The other halves ofthe implants, layered with titanium hydride-amoxicillin, may be analyzedin the bacterial growth assay according to Example 5. Identical controlimplants present is the same electrolytic cells as the experimentalimplants but not connected to the cathode may be used as controls.

Example 11

Preparation of a Osteoinductive Titanium Hydride Implant Surface LayerContaining a Biomolecule

Implants prepared as in Example 1 (titanium hydride-amelogenin) areplaced in calibrated bone defects in the tibia bone of rabbits, makingsure that fenestrations into the bone marrow beneath the implants allowmigration of osteogenic cells to the modified implant surfaces, using astandardized and validated model (Rønold and Ellingsen, European Societyfor Biomaterials Conference, Amsterdam, October 2000). On the day aftersurgery and every following week the rabbits are given an intravenouscalcein (Sigma) injection of 10 mg/kg body weight. At four weeks afterplacing of the modified implants and control implants the rabbits willbe sacrificed and the tibia removed, fixed in 4% formaldehyde andembedded for preparation of ground sections through the bone and theintegrated implant material. Identical control implants present is thesame electrolytic cell as the experimental implants but not connected tothe cathode may be used as controls.

1. A method for preparing a medical prosthetic device or medicalimplant, said method comprising: subjecting surface parts of a metalmaterial selected from the group comprising titanium or an alloythereof, zirconium or an alloy thereof, tantalum or an alloy thereof,hafnium or an alloy thereof, niobuim or an alloy thereof and achromium-vanadium alloy to an electrolysis treatment in the presence ofone or more biomolecule substances, said metal material constitutingcathode during said electrolysis.
 2. The method as claimed in claim 1,wherein a layer of a corresponding hydride material selected from thegroup comprising titanium hydride, zirconium hydride, tantalum hydride,hafnium hydride, niobium hydride and chromium and/or vanadium hydride isformed on said surface parts and the biomolecule(s) become associatedwith the hydride material.
 3. The method as claimed in claim 1, whereinthe electrolysis treatment is carried out in the presence of anelectrolyte comprising the biomolecule substance.
 4. The method asclaimed in claim 1, wherein said medical prosthetic device or implant isa dental implant.
 5. The method as claimed in claim 1, wherein saidmetal material is titanium.
 6. The method according to claim 1, whereinthe biomolecule is non-covalently associated with the device or implant.7. The method according to claim 1, wherein said electrolysis treatmentis carried out at a pH between 0 and
 10. 8. The method according toclaim 1, wherein said electrolysis treatment is carried out at a pHbetween 2 and
 5. 9. The method according to claim 1, wherein saidelectrolysis treatment is carried out in the presence of an electrolytehaving an ionic strength between 0.01M and 10 M.
 10. The methodaccording to claim 1, wherein said electrolysis treatment is carried outat a temperature between 0° C. and 100° C.
 11. The method according toclaim 1, wherein said electrolysis treatment is carried out at atemperature between 20° C. and 80° C.
 12. The method according to claim1, wherein the concentration of biomolecule substance in the electrolyteis between about 1 picogram per ml and 50 milligrams per ml.
 13. Themethod according to claim 1, wherein the electrolysis treatment iscarried out at a voltage between 0.1 volts and 1000 volts.
 14. Themethod according to claim 1, wherein the electrolysis treatment iscarried out at a voltage below 10 volts.
 15. The method as claimed in 1,wherein the electrolysis treatment is carried out at a current densitybetween about 0.1 mA per cm² and 1A per cm².
 16. The method as claimedin 1, wherein the electrolysis treatment is carried out at a currentdensity of about 1 mA/cm².
 17. The method as claimed in claim 1, whereinthe electrolysis treatment is carried out for between about 0.5 and 24hours.
 18. The method as claimed in claim 1, wherein the electrolysistreatment is carried out under aseptic or sterile conditions.
 19. Themethod as claimed in claim 1, wherein the electrolysis treatment iscarried out in the presence of an electrolyte comprising the biomedicalsubstance with a net positive charge.
 20. The method as claimed in claim1, wherein the biomolecule substance exhibits a net positive chargedissolved in a salt solution having an ionic strength within the rangefrom 0.01 to 10 M, a temperature within the range from 0 to 100° C., anda pH within the range from 0 to
 10. 21. The method as claimed in claim1, wherein the biomolecule substance is an ampholyte.
 22. The method asclaimed in claim 1, wherein the biomolecule substance is selected fromthe group comprising substances consisting of natural, recombinant orsynthetic bio-adhesives; natural, recombinant or synthetic cellattachment factors; natural, recombinant or synthetic biopolymers;natural, recombinant or synthetic blood proteins; natural, recombinantor synthetic enzymes; natural, recombinant or synthetic extracellularmatrix proteins; natural, recombinant or synthetic extracellular matrixbiomolecules; natural, recombinant or synthetic growth factors; natural,recombinant or synthetic hormones; natural, recombinant or syntheticpeptide hormones; natural, recombinant or synthetic deoxyribonucleicacids (DNA); natural, recombinant or synthetic ribonucleic acids (RNA);natural, recombinant or synthetic receptors; natural, recombinant orsynthetic enzyme inhibitors; drugs; biologically active anions andcations; natural, recombinant or synthetic peptides; natural,recombinant or synthetic proteins; natural or synthetic vitamins;adenosine monophosphate (AMP); adenosine diphosphate (ADP); adenosinetriphosphate (ATP); marker biomolecules; amino acids; fatty acids;nucleosides; nucleotides (RNA and DNA bases); and sugars.
 23. The methodas claimed in claim 1, wherein the biomolecule substance is selectedfrom the group comprising substances consisting of natural, recombinantor synthetic bio-adhesives; natural, recombinant or synthetic cellattachment factors; natural, recombinant or synthetic biopolymers;natural, recombinant or synthetic blood proteins; natural, recombinantor synthetic enzymes; natural, recombinant or synthetic extracellularmatrix proteins; natural, recombinant or synthetic extracellular matrixbiomolecules; natural, recombinant or synthetic growth factors; natural,recombinant or synthetic hormones; natural, recombinant or syntheticpeptide hormones; natural, recombinant or synthetic deoxyribonucleicacids (DNA); natural, recombinant or synthetic ribonucleic acids (RNA);natural, recombinant or synthetic receptors; natural, recombinant orsynthetic enzyme inhibitors; natural, recombinant or synthetic peptides;natural, recombinant or synthetic proteins; natural or syntheticvitamins; adenosine monophosphate (AMP); adenosine diphosphate (ADP);adenosine triphosphate (ATP); amino acids; fatty acids; nucleosides;nucleotides (RNA and DNA bases); and sugars.
 24. The method as claimedin claim 1, wherein the biomolecule substance is a drug.
 25. The methodas claimed in claim 24, wherein the biomolecule is a statin.